WO2005117940A2 - Cell death modulation via antagonists of fasl and fas activation - Google Patents
Cell death modulation via antagonists of fasl and fas activation Download PDFInfo
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- WO2005117940A2 WO2005117940A2 PCT/US2005/014122 US2005014122W WO2005117940A2 WO 2005117940 A2 WO2005117940 A2 WO 2005117940A2 US 2005014122 W US2005014122 W US 2005014122W WO 2005117940 A2 WO2005117940 A2 WO 2005117940A2
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C07K—PEPTIDES
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
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- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions
- Programmed cell death is a cell suicide process that occurs in animal cells. Normally, cell death takes place during the growth and development of an organism, and it is used to eliminate excess cells. Cell death also is triggered when cells become damaged beyond normal repair mechanisms, or dangerous to the organism, such as autoimmune cells. Suppression, overexpression, or mutation of the genes that control the process, however, can lead to disease. More specifically, excessive cell death activity has been linked to liver disease, kidney disease, disorders of the pancreas, autoimmune diseases such as AIDS and rheumatoid arthritis, and neurodegenerative disorders such as Alzheimer's or Parkinson's. The ability to control and suppress overactive cell death would be a useful tool in the treatment of many serious diseases.
- Fas is a transmembrane cell surface receptor expressed in a variety of tissues and cell types and is activated by the binding of its cognate ligand known as Fas-Ligand (FasL).
- Fas is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily of apoptosis promoting cell surface transmembrane receptors.
- TNFR Tumor Necrosis Factor Receptor
- the binding of FasL to Fas results in receptor self-trimerization and clustering, which subsequently engages a cascade of biochemical events that culminates in apoptosis.
- the Fas/FasL system has been linked to human diseases, including liver and autoimmune diseases.
- Met a transmembrane cell surface receptor for hepatocyte growth factor, has the ability to bind the Fas receptor and prevent its activation.
- Met is a heterodimer consisting of an alpha and a beta subunit linked together by a disulfide bond.
- the alpha chain is approximately 282 amino acids in length (amino acid residues 25-307) and remains entirely extracellular, while the beta chain is a single-pass transmembrane protein measuring approximately 1080 amino acid residues long (of which amino acids 308 to 932 remain extracellular) and harbors the tyrosine kinase and the associated signaling and regulatory domains within its intracellular cytoplasmic region.
- the invention provides a polypeptide that attenuates the activation of one or more members of the TNFR superfamily particularly, Fas, TNFRl, or both.
- the inventive polypeptide can be used for preventing activation of the cell death pathway and can be used therapeutically in treating conditions, such as those described above, associated with aberrant activation of the cell death pathway by Fas or TNFRl .
- the invention also provides a pharmaceutical composition comprising the inventive polypeptide and a pharmaceutically acceptable carrier, which can be administered to facilitate treatment of such conditions.
- Figures la-lg present data demonstrating that the alpha subunit of Met (AlphaMet) interacting with the extracellular part of Fas via novel motif YLGA (SEQ ID NO:l).
- the Met alpha but not beta chain binds to Fas (Fig. la).
- Various regions of the mouse Met cDNA corresponding to the alpha chain or the extra-cellular portion of the Met beta chain were cloned into expression vectors (pCRT7 TOPO/AlphaMet or pCRT7 TOPO/beta chain) and subjected to in vitro transcription/translation by a rabbit reticolucyte lysate translation system. Empty vector was included in each experiment as a control.
- the expressed proteins were labeled with 35 S-Methionine.
- Pull down assays were performed using Fas-Fc, a chimeric molecule which lacks the interacellular cytoplasmic domain and protein-A agarose as described in Wang et al., 2002.
- Figure lb is a schematic presentation of the Met alpha chain and its mutant version used to map the region interacting with Fas.
- the C-terminus of each construct contains a hexa-Histidine tag for ease of detection. Start cordon ATG was added to the N-terminally truncated constructs.
- the YLGA (SEQ ID NO:l) motif mutation was prepared by PCR, in which YLGA (SEQ ID NO: 1) was replaced by DHER (SEQ ID NO:24).
- Figure lc presents data demonstrating that the N-terminal 100 amino acids of the Met alpha subunit are sufficient to bind Fas.
- In vitro translated 35 S-methionine labeled truncated/mutated AlphaMet proteins were pulled down with 2 ⁇ g of Fas-Fc or the same amount of IgG.
- Lane 1 10% of AlphaMet input; Lane 2: AlphaMet with IgG control; Lanes 3 to 8: pull down of various truncated AlphaMet proteins; N-terminal 1-106 residues; C- terminal truncated, residues 1-210; alpha chain, residues 1-306; alpha chain with transmembrane domain, 1-338; N-terminally truncated alpha chain, residues 100-306; and N- terminally truncated with addition of the transmembrane domain 100-338, respectively.
- Figure Id depicts the amino acid sequence alignment of alpha chain with FasL, determined using the CLUSTALW, SIM+LALNVIEW, and ALIGN at ExPASy Proteomic Tools (available on the internet at us.expresly.org/tools). The numbers indicate the amino acid residues of the indicated protein.
- Figure le presents data demonstrating that wild type AlphaMet but not the YLGA mutant binds to Fas. Recombinant His-tagged AlphaMet or its mutant version were expressed in E. coli, purified with chromatography, and subjected to Fas-Fc (lanes labeled Fas) or control IgG pull down experiments, and detected by Western blot using anti-His antibody that recognizes C-terminal His.
- FIG. 1 g presents data demonstrating that synthetic peptides derived from mouse or human AlphaMet bind to Fas with high affinity as determined by ELISA.
- Peptides (1 ⁇ g/ml) were coated into the 96- well microtiter plates in triplicate and after blocking with 1% BSA, increasing amounts of Fas-Fc were added as indicated.
- the control peptide was a scrambled 13mer.
- anti-human IgM-HRP conjugates (1 :20,000) were added to detect the bound Fas-Fc using TMD dye and the OD value was determined at a wavelength of 450nm.
- One-way ANOVA was carried out for determination of statistical significance.
- Figures 2a-2e present data demonstrating that AlphaMet competes with Fas-ligand for Fas binding.
- Figure 2a presents data demonstrating that recombinant purified His-tagged AlphaMet (approximately 5 ⁇ g) incubated with Fas-Fc in the presence or absence of FasL or unrelated protein (albumin, Fig. 2c), subjected to pull down experiments followed by Western blot using anti-His antibody that recognizes C-terminal His. This antibody also recognizes Fas-Fc which is also tagged at the C-terminus by His.
- Figure 2b a fixed amount of purified recombinant Fas-ligand was added to Fas-Fc in the absence or presence of increasing amounts of AlphaMet as indicated.
- FIG. 2d shows the results of ELISA, carried out as described above.
- Fas ligand (1 ⁇ g/ml) was coated in the wells of a microtiter plate (triplicate wells) then 1 ⁇ g/ml of Fas-Fc was added in the absence or presence of an increasing amount of the indicated polypeptide.
- a 13mer YLGA polypeptide derived from FasL also was synthesized and used. The control peptide was a scrambled 13mer.
- Anti human IgM-HRP conjugates were used to detect the bound Fas-Fc.
- Figure 2e presents data demonstrating that AlphaMet and YLGA polypeptides inhibit FasL binding to Fas in Jurkat cells.
- Cells (3X10 5 ) were plated in 24- well plates having Poly-L-Lysine pre-treated cover glass. 2 ⁇ g/ml of Fas ligand was added and the cells were incubated for 30 min on ice. Cells were then washed by PBS twice, and were subjected to immunostaining using anti-Fas ligand antibody and fluorescent microscopy.
- Fas ligand was not added, in B, only Fas ligand was added, in C-F, Fas ligand was added with different polypeptides as follows: AlphaMet, 12mer YLGA, control peptide, and YLGA mutated AlphaMet, respectively.
- Figures 3a-3f present data demonstrating that AlphaMet prohibits Fas homo- trimerization and Fas microaggregation.
- Figure 3 a presents data collected from Jurkat cells (1X10 6 ) treated with 2 ⁇ g/ml of Fas Ligand with or without AlphaMet for 30 minutes on ice. Cells were washed with PBS twice and then incubated with 2mM of DTSSP in PBS for 30 minutes. The crosslinking process was stopped by 20mM of Tris (pH 7.5) for another 30 minutes. Cells were spun down and washed with PBS twice and lyzed in RIPA buffer.
- FIG.3b Cell lysates were subjected to SDS-PAGE under non-reducing or reducing conditions (Fig.3b) and Western blot analysis using the mouse monoclonal antibody against human Fas. Fas monomer migrating as a doublet as well as trimerized Fas are indicated in the figure.
- Figure 3c depicts the quantification of the data in Fig. 3a by densitometry. The data are the mean and SE from three different independent crosslinking and Western blot experiments.
- Figure 3d shows a FRET analyses of Hepa 1-6 transfected with Fas- fluorescent protein chimeric proteins (CFP-Fas and YFP-Fas).
- Frame A Acceptor CFP-Fas image
- Frame B Acceptor pre-bleach CFP-Fas image
- Frame C Acceptor post-bleach image
- Frame D Donor YFP- Fas image
- Frame E Donor pre-bleach image
- Frame F Donor pre-bleach image.
- the CFP- Fas strength in the selected region obtained a gain of 19.3% efficiency in the presence of FasL for 15 minutes. No FRET was observed without FasL. In the presence of FasL and AlphaMet, no significant FRET could be detected.
- Figure 3e depicts the results of experiments in which CFP-tagged Fas or CFP control vector were transiently expressed in Hepa 1-6 Cells (3X10 5 ).
- Cells were treated with 1 ⁇ g/ml of Fas ligand or lOOng/ml of Jo2 with or without 5 ⁇ g/ml of AlphaMet or 1 ⁇ g/ml of YLGA 12mer for 30 minutes on ice. Cells were fixed and analyzed by fluorescence microscopy. Frame A: AlphaMet treatment only, no FasL; Frame B: Fas ligand treatment only; Frame C: Fas ligand and AlphaMet treatment; Frame D: Fas ligand and 12mer YLGA; Frame E: Jo2 treatment only and Frame F: Jo2 and AlphaMet treatment. The percentage cells demonstrating capping of Fas was calculated by enumerating at least 300 cells from random fields and the data were quantified and depicted graphically in Figure 3f.
- FIGs 4a-4d present data demonstrating that AlphaMet and YLGA polypeptide attenuate apoptosis in Jurkat and Hepa 1-6 cell lines.
- Jurkat cells 1.5X10 5
- apoptosis was determined by Trypan Blue staining using a hemocytometer. The data are representative and were repeated at least four times.
- Figure 4b presents data demonstrating that cleaved Caspase 3 fragment was detected in apoptotic Jurkat cells by specific monoclonal antibody.
- Figures 5a-5c presents the results of an experiment demonstrating protection of the liver by AlphaMet and the corresponding polypeptides from Fas-induced apoptosis in Balb/c Mice.
- Balb/c mice (10 per group) were injected with Jo2 (2.5 ⁇ g/g of mouse body weight) in the presence of AlphaMet or polypeptides as indicated, and analyzed for the extent of hepatocyte apoptosis and liver damage by immunohisological and biochemical techniques.
- the percentage of TUNEL positive was determined in multiple liver lobes of each liver in random fields counting at least 500 hepatocytes in a double-blind manner.
- FIG. 5b graphically presents the quantification of hepatocyte apoptosis TUNEL positive nuclei.
- the data were analyzed by One-way ANOVA and P ⁇ 0.05 was considered significant.
- Figure 5c an equal amount of livers were subjected to Caspase 3 activity assay using DEVD-AFC as a substrate and DEVD-CHO as an inhibitor, as recommended by the manufacturer. The data are the mean and SE and were repeated in three independent experiments. The data were analyzed by One-way ANOVA and PO.05 was considered significant.
- the invention provides a synthetic polypeptide that attenuates the activation of a member of the TNFR superfamily, which includes Fas, TNFRl, and other proteins. More preferably, the polypeptide attenuates the activation of both Fas and TNFRl.
- the inventive polypeptide attenuates the activation of Fas, TNFRl, or other members of the TNFR superfamily in that it reduces the activation of such protein to a measurable extent. Attenuation can be assessed using in vitro assays (e.g., pull down experiments as described below, immunohistological techniques such as ELISA, Western blotting, immunoprecipitation, etc.).
- Fas Activation of Fas, TNFRl, or both (or other members of the TNFR superfamily) can also be attenuated in vivo.
- a cell line can be transfected with a plasmid containing the polypeptide. Apoptosis can then be induced by the addition of FasL, followed by a flow cytometry analysis of the cells.
- the inventive polypeptide can attenuate the activation of the members of the TNFR superfamily (e.g., Fas, TNFRl, or both) to a varying degree, depending on which member of the TNFR superfamily is to be affected and also on the exact sequence of the polypeptide. Desirably, however, the inventive polypeptide attenuates activation of the members of the TNFR superfamily to a degree sufficient to modulate apoptosis. In preferred embodiments, the inventive polypeptide blocks or substantially blocks activation of, for example, Fas, TNFRl, and preferably both.
- the inventive polypeptide attenuates the activity of members of the TNFR superfamily (especially Fas, TNFRl, or both) by at least about 70%, such as at least about 80% or even at least about 90%).
- the inventive polypeptide blocks or substantially blocks the activation of the member of the TNFR superfamily, such as, for example, attenuating the activation of Fas, TNFRl, or both (or other member of the TNFR superfamily) by at least about 90% or even above about 98% or 99%.
- the inventive polypeptide comprises at least four or at least five or at least six, such as at least twelve or at least about 36 amino acids.
- inventive polypeptide can also contain up to several hundred amino acids, such as having up to about 100 or up to about 200 or up to about 300 amino acids.
- inventive polypeptide can include longer amino acid sequences than these, but such is not typical.
- inventive polypeptide can comprise, consist of, or consist essentially of the alpha domain of Met, or C-terminal truncations of AlphaMet, the sequence of which is known (Stamos et al., 2004). Three examples of such truncations comprise amino acids 1-106, amino acids 1-210, or amino acids 1-306.
- inventive polypeptide is, however, a synthetic sequence in that it does not include the entire sequence of AlphaMet amino acids (indeed, the three examples just listed are C- terminal deletions).
- the inventive polypeptide also can comprise a truncation of a Semaphorin and Plexin.
- the YLGAV (SEQ ID NO:5) corresponding to PlexinA4 is identical to the YLGAV (SEQ ID NO:5) present in the Fas binding domain of FasL.
- FLGAV (SEQ ID NO:37) the human counterpart of YLGAV (SEQ ID NO:5) also is very active in binding to Fas and inhibiting FasL binding to Fas and hence inhibiting Fas activation.
- the inventive polypeptide is a shorter polypeptide, such as consisting of from about 5 to about 50 amino acids (e.g., less than about 15, and preferably less than about 12 or less than about 10 amino acids; or between about 4-6 amino acids, about 10-15 amino acids, about 15-20 amino acids, about 20-25 amino acids, about 25-30 amino acids, about 30- 35 amino acids, or about 30-40 amino acids).
- amino acids e.g., less than about 15, and preferably less than about 12 or less than about 10 amino acids; or between about 4-6 amino acids, about 10-15 amino acids, about 15-20 amino acids, about 20-25 amino acids, about 25-30 amino acids, about 30- 35 amino acids, or about 30-40 amino acids).
- Preferred examples of the inventive polypeptide comprise, consist of, or consist essentially of the 4 contiguous amino acid sequences YLGA (SEQ ID NO:l), FLGA (SEQ ID NO: 32), YLGG (SEQ ID NO:2), or FLGG (SEQ ID NO:34).
- the inventive polypeptide comprise, consist of, or consist essentially of the following 5 contiguous amino acid residues having the sequence IYLGA (SEQ ID NO:3), IYLGG (SEQ ID NO:4), YLGAV (SEQ ID NO:5), YLGGV (SEQ ID NO:6), IFLGA (SEQ ID NO:35), IFLGG (SEQ ID NO:36), FLGAV (SEQ ID NO:37), or FLGGV (SEQ ID NO:38), or the following 6 contiguous amino acid residues having the sequence IYLGAV (SEQ ID NO:7), IYLGGV (SEQ ID NO:8), IFLGAV (SEQ ID NO:39), or IFLGGV (SEQ ID NO:40).
- inventive polypeptide is not limited to the amino acid sequences described above and can include conservative substitutions or other changes that retain the ability of the inventive polypeptide to attenuate the activation of the member of the TNFR superfamily (e.g., Fas or TNFRl, or both).
- TNFR superfamily e.g., Fas or TNFRl, or both.
- inventive polypeptide can be prepared by methods known to those of ordinary skill in the art.
- inventive polypeptide can be synthesized using solid phase polypeptide synthesis techniques (e.g. Fmoc).
- the polypeptide can be synthesized using recombinant DNA technology (e.g., using bacterial or eukaryotic expression systems).
- the invention provides genetic vectors (e.g., plasmids) comprising a sequence encoding the inventive polypeptide, as well as host cells comprising such vectors.
- inventive polypeptide in recombinant form e.g., plasmids
- the inventive polypeptide can be isolated and/or purified (or substantially isolated and/or substantially purified). Accordingly, the invention provides the inventive polypeptide in substantially isolated form.
- the polypeptide can be isolated from other polypeptides as a result of solid phase protein synthesis, for example.
- the polypeptide can be substantially isolated from other proteins after cell lysis from recombinant production. Standard methods of protein purification (e.g., HPLC) can be employed to substantially purify the inventive polypeptide.
- the invention provides a preparation of the inventive polypeptide in a number of formulations, depending on the desired use.
- the polypeptide can be formulated in a suitable medium solution for storage (e.g., under refrigerated conditions or under frozen conditions).
- suitable medium solution for storage e.g., under refrigerated conditions or under frozen conditions.
- Such preparations can contain protective agents, such as buffers, preservatives, cryprotectants (e.g., sugars such as trehalose), etc.
- the form of such preparations can be solutions, gels, etc., and the inventive polypeptide can, in some embodiments, be prepared in lyophilized form.
- such preparations can include other desired agents, such as small molecules or even other polypeptides and proteins, if desired.
- the invention provides such a preparation comprising a mixture of different embodiments of the inventive polypeptide (e.g., a plurality of polypeptide species as described herein).
- the invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising of one or more of the inventive polypeptides (including mixtures thereof) and a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier Any carrier which can supply the polypeptide without destroying the vector within the carrier is a suitable carrier, and such carriers are well known in the art.
- the composition can be formulated for parenteral, oral, or topical administration.
- a parenteral formulation could consist of a prompt or sustained release liquid preparation, dry powder, emulsion, suspension, or any other standard formulation.
- An oral formulation of the pharmaceutical composition could be, for example, a liquid solution, such as an effective amount of the composition dissolved in diluents (e.g., water, saline, juice, etc.), suspensions in an appropriate liquid, or suitable emulsions.
- diluents e.g., water, saline, juice, etc.
- An oral formulation could also be delivered in tablet form, and could include excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients.
- a topical formulation could include compounds to enhance absorption or penetration of the active ingredient through the skin or other affected areas, such as dimethylsulfoxide and related analogs.
- the pharmaceutical composition could also be delivered topically using a transdermal device, such as a patch, which could include the composition in a suitable solvent system with an adhesive system, such as an acrylic emulsion, and a polyester patch.
- a transdermal device such as a patch
- an adhesive system such as an acrylic emulsion
- the invention also provides a method of employing the inventive polypeptide to attenuate the activation of one or more members of the TNFR superfamily, desirably Fas and/or TNFRl.
- Such method can be employed, for example, to inhibit cell death (e.g., apoptosis) or an inflammatory response in cells and tissues, and it can be employed in vivo, ex vivo or in vitro.
- the invention provides for the use of the inventive polypeptide for attenuating cell death in accordance with such methods.
- the inventive polypeptide is provided to cells, typically a population of cells (e.g., within a suitable preparation, such as a buffered solution) in an amount and over a time course sufficient to inhibit apoptosis within the cells or to inhibit inflammation. If desired, a controlled population untreated with the inventive polypeptide can be observed to confirm the effect of the inventive polypeptide in reducing the inhibition of cell death or inflammation within a like population of cells.
- the inventive polypeptide can be delivered to a human or animal subject in an amount and at a location sufficient to inhibit or attenuate apoptosis or inflammation within the patient (e.g., within desired tissue).
- the inventive polypeptide can be formulated into a suitable pharmaceutical composition (e.g., as described above or as otherwise known to those of ordinary skill in the art) for delivery into the subject.
- the delivery can be local (e.g., by injection or implantation within the desired tissue to be treated) or systemic (e.g., by intravenous or parenteral injection).
- systemic delivery of the inventive polypeptide can be employed to attenuate apoptosis in liver tissue.
- the inventive polypeptide can be employed (alone or adjunctively with other treatments) to treat diseases or disorders of the liver, such as liver failure or hepatitis, in patients in need of such treatment.
- the inventive polypeptide also can be used (alone or in conjunction with other modes of treatment) to treat patients suffering from other diseases or disorders mediated by cell death or apoptosis and tissue inflammation, such as liver disease, kidney and lung diseases, disorders of the pancreas, autoimmune diseases such as AIDS and rheumatoid arthritis, and neurodegenerative disorders such as Alzheimer's, Parkinson's or spinal cord injury.
- the invention provides a method for treating patients suffering from such diseases or disorders and in need of treatment.
- a pharmaceutical composition comprising at least one species of inventive polypeptide is delivered to such a patient in an amount and at a location sufficient to treat the disorder or disease.
- the invention also provides for the use of the inventive polypeptide for preparing a medicament to treat such disorders.
- the inventive polypeptide or pharmaceutical composition comprising such
- inventive method of treating a patient most preferably substantially alleviates or even eliminates such symptoms; however, as with many medical treatments, application of the inventive method is deemed successful if, during, following, or otherwise as a result of the inventive method, the symptoms of the disease or disorder in the patient subside to a degree ascertainable.
- the experimental data reveal that polypeptides having a sequence similar to that of the conserved region of AlphaMet, FasL, Met related family members such as Plexin 4A, or the TNF family members are able to block the activation of Fas or TNFRl by preventing the receptors from self-trimerization, clustering, and capping.
- these examples demonstrate that the binding of Fas to FasL is abrogated by increasing amounts of polypeptide corresponding to the YLGA (SEQ ID NO:l) or FLGA (SEQ ID NO:32) motif of AlphaMet, or the YLGA (SEQ ID NO:l) motif of FasL.
- polypeptides of the invention suppress the ability of Fas to self-trimerize and cluster.
- Anti-Fas antibodies, anti-Fas ligand antibody, anti- Met antibodies, and Protein A Agarose beads were from Santa Cruz and UpState Biotechnology. Anti-C-terminal His-tag antibody was from Invitrogen. Fas-Fc chimera protein, soluble Fas Ligand, and its crosslinking antibody were purchased from R&D. Anti- human IgM-HRP conjugates were from Chemicon. Chemical crosslinker 3,3'dithiobis [sulfosuccinimidylpropionate] (DTSSP), ELISA color reagent 3, 3', 5, 5' Tetramethylbenzidine (TMB), Lyzozyme, protease inhibitor cocktail, and DEAE column were from Sigma.
- Nickel- beads for protein purification were from Invitrogen. Isotopes S labeled methionine and Cystine were from Amersham Biosciences. Synthetic polypeptides were made by Genemed Synthesis, Inc., CA. All other chemicals used in the experiments were commercially available with the highest quality.
- Mouse origin MET cDNA was used as a PCR template, and a variety of forward (included ATG start codon) and reverse primers were designed based on the mouse C-MET gene sequence: Forward 1, 5'-ATGAAGGCTCCCACCGTG (SEQ ID NO:9); ForwardlOO, 5'-ATGGGGACTGCAGCAGCAAAG (SEQ ID NO:10); Forward307, 5'-ATGCCACAAGGGAAGAAGTG (SEQ ID NO:l 1); ReverselO ⁇ , 5'- CTTTGCTGCTGCAGTCCCG (SEQ ID NO:12); Reverse210, 5'- CAGTGAATAACCAGGAGG (SEQ ID NO: 13); Reverse306, TCTCTTCCTT CTTTTTTCTGTCAG (SEQ ID NO: 14); Reverse929, 5'-ATTCTGATCCGGTTGAACG (SEQ ID NO: 15).
- YLGA was mutated by PCR, with the BamH I overhang site primers as follows: Forwardl, 5'-ATGAAGGCTCCCACCGTG (SEQ ID NO: 16); Reverse YLGA 5'- CGGGATCGATCACGAACGCACAAACTACATTTATG (SEQ ID NO: 17); Forward YLGA, 5'-CGGGGATCCATGGCCGTGTAGGACGACATTCTGG (SEQ ID NO: 18); ReverselO ⁇ , 5'-CTTTGCTGCTGCAGTCCCG (SEQ ID NO:19). Two PCR fragments were digested with BamH I and ligated into one fragment.
- PCR products were inserted into the pCRT7 TOPO vector (Invitrogen), and the DNA inserts were confirmed by DNA sequencing.
- C-terminal tagged polyhistidine (6xHis) DNA constructs were produced by PCR using pCRT7 TOPO inserts as templates.
- Jurkat cells were cultured overnight at a density of 10 6 per ml in the 24-well plates that contained cover glass (pretreated with 0.1% of poly-L-Lysine solution). Cells were treated with Fas ligand with or without AlphaMet, or the synthesized polypeptides at 4°C for 30 minutes. Cells were then washed with PBS pH 7.4 twice, fixed with cold methanol for 15 minutes, and blocked by incubating with blocking solution (5% donkey serum in PBS) for 1 hour. Anti-Fas ligand antibody (at a concentration of 1 : 1000) was diluted in blocking buffer, and incubated with cells for one additional hour.
- Apoptosis assay Pre- washed Jurkat cells in RPMI 1640 medium containing 1% FBS were inoculated in 96-wells at a density of 10 5 cells per well. Apoptosis was induced by cross-linked human Fas-ligand (R&D Systems) at a concentration of 0.5ng/ml or Anti- Fas(human, activating) clone CHI 1 (Upstate biotechnology) at a concentration of 500 ng /ml. Hepa 1-6 cells were overnight starved before induction of apoptosis.
- the apoptosis in Hepa 1 -6 was induced by anti-mouse Fas antibody Jo2 (200ng/ml) or human recombinant Fas- ligand.
- the extent of cell viability and apoptosis was determined by several methods such as Trypan Blue stain, flow cytometry, LDH assay, and Hoechst staining.
- Hepa 1-6 cell transfection pCR3.1 /inserts plasmids were transfected into Hepa 1- 6 cells by the lipofectamine method (Invitrogen). Hepa 1-6 cells were plated into 6- well plates until the cells were 80% confluent, 2 ⁇ g of plasmid DNA was premixed with lipofectamine in a volume of 200 ⁇ l for 30min, then the mixture was added into the pre- washed cells with 0.8ml of serum-free medium. After the cells were cultivated at 5% CO 2 , 37°C for 5hours, FBS was added in a final concentration of 10%. Transient transfected cells were lysed by RIPA buffer. The cell lysate were subjected to Western blotting, immunoprecipitation, and apoptosis.
- Statistical Analysis Analysis of Variance (One-way ANOVA) and the Student t- test were utilized for determining the statistical significance of the data.
- ELISA Synthesized polypeptides (Genemed Synthesis, Inc. South San Francisco, CA) were dissolved in water and diluted in the coating buffer (0.05M of Sodium Carbonate, 0.02% of Sodium Azide, pH 9.6) to a final concentration of lug/ml. One hundred microliter aliquots were added to coat each well of a 96-well microtitre plate. The coated plates were then washed with PBS-Tween (0.5%) three times. The plates were blotted and dried by tapping upside down on tissue paper.
- the CFP and YFP-tagged Fas mammalian expression vectors and their counterpart control vectors were a generous gift from Dr. Richard Siegel at NIH, Bethesda. These vectors have been previously used by Siegel et al., 2000, in FRET studies to demonstrate Fas self-assembly.
- Hepa 1-6 Cells were transiently transfected with one or two of these plasmids in combination or with control CYP/CFP vector lacking Fas molecule by o the previously described method. After overnight culture, the transfected cells were treated with Fas ligand with or without AlphaMet. Cells were fixed by cold Methanol and applied to Fret analysis. Imaging was performed with Leica Confocal systems.
- FRET imaging acquisition and data analysis were performed with LCS Microlab software.
- Pre- and post- bleaching image sets of both donor (CFP) and acceptor (YFP) were acquired at the laser wave length of 473nm or 517nm correspondingly.
- CFP signals outlining the cell membrane were selected as the region of interest.
- Fret efficiency (E) was calculated as Dpr e )/D P ost (Dpost and D pre are the mean emission intensity prior to and following YFP bleaching).
- the alpha and the beta subunit expression vectors encoding the alpha chain (amino acids residues 1 to 306) or the extracellular region of Met beta chain (amino acids 308 to 968 which also contains the transmembrane portion) were analyzed to determine whether both were involved in the interaction with Fas. As shown in Fig. la, only Met alpha chain but not the Met beta chain region bound specifically to the Fas molecule (Fig. la, left and the right panels, respectively).
- the alpha subunit of Met was further analyzed to determine which part of the AlphaMet was crucial for Fas binding.
- Several N-terminally or C-terminally truncated AlphaMet expression vectors tagged with His-tag were generated (see Fig. lb) and tested in similar fashion.
- Fig. lc lane 5 the full length AlphaMet bound Fas very efficiently.
- C-terminally truncated AlphaMet constructs (amino acid residues 1 to 106 and 1 to 210) also showed robust Fas binding (Fig. lc, lanes 3 and 4).
- N-terminally truncated AlphaMet lacking the first 100 amino acid residues exhibited no detectable Fas binding (Fig. lc lane 7).
- the YLGA motif was mutated to positively or negatively charged amino acids DHER in the context of the full length AlphaMet (see Fig.1 b), expressed and purified in E. coli, and pull down assays were performed using the Fas-Fc chimeric construct (Fig. le).
- YLGA mutated AlphaMet totally lost its ability to bind to Fas, whereas the wild type AlphaMet bound avidly (Fig. le).
- the N-terminally truncated AlphaMet having residues lacking the first 100 amino acid residues did not bind to Fas.
- the wild type, YLGA-mutated full length AlphaMet (residues 1 to 306) and the N-terminally deleted AlphaMet (having residues 100 to 306) were directly added to Jurkat cells and Hepa 1-6 cells which express high levels of endogenous Fas. Immunoprecipitation was then performed using anti-Fas antibody or control IgG followed by Western blot analysis using anti-His-tagged antibody as a probe. As predicted, only wild type but not the mutant AlphaMet could be co-precipitated with anti-Fas (Fig. If and g).
- Fas-Fc was coated into the wells of a 96-well plate and increasing amounts of coated polypeptides were quantified. Initially peptides of 36mer (FTAETPIQNVVLHGHHIYLGATNYIYVLNDKDLQKV, SEQ ID NO:25), 12mer (HHIYLGATNYIY, SEQ ID NO:26), and 4mer (YLGA, SEQ ID NO:l) in length were tested. These polypeptides bound very specifically and efficiently to Fas although the 4mer YLGA (SEQ ID NO:l) had reduced activity (Fig.lh). Additional 4mer polypeptides were synthesized in which Y, L, or G was replaced by R (i.e.
- RLGA SEQ ID NO:27
- YRGA SEQ ID NO:28
- YLRA SEQ ID NO:29
- a conservative substitution like Y to F FLGA SEQ ID NO:32
- AHSSYLGAVFNLT SEQ ID NO:30
- Example 2 This example demonstrates that AlphaMet competes with FasL for binding to Fas.
- AlphaMet could compete with FasL for binding to Fas.
- Binding and pull down assays were performed using Fas-Fc, recombinant full length AlphaMet, and recombinant FasL.
- Fig. 2a and 2c the binding of AlphaMet to Fas was completely abrogated by increasing amounts of FasL but not unrelated proteins, respectively.
- the addition of increasing amounts of AlphaMet blocked the FasL binding to Fas (Fig. 2b).
- Jurkat cells were treated with 1 ⁇ g/ml of human recombinant FasL at 4°C for 30 minutes in the presence or absence of recombinant wild type or YLGA mutated AlphaMet or synthetic polypeptides as indicated. The cells were fixed and stained for FasL using anti-FasL antibody.
- Example 3 demonstrates that the inventive polypeptides suppress Fas molecule assembly on the cell membrane.
- Fas mainly exists in monomeric form in hepatic and Jurkat cells (Fig.3a). It was possible that Fas trimerization process would be affected by AlphaMet. Chemical crosslinking experiments were performed on Jurkat cells and a Western blot using anti-Fas antibody to detect Fas trimerization status was utilized to detect Fas. Thiol-cleavable, membrane-impermeant chemical cross-linker 3,3'dithiobis [sulfosuccinimidylpropionate] (DTSSP) was then applied.
- DTSSP dithiobis
- Fas-Ligand a specific high molecular mass (Mr of about 160) complex was found in Jurkat cells which increased about 3 fold in intensity as compared to the ligand unstimulated crosslinked cells (Fig. 3a and 3c).
- Mr specific high molecular mass
- AlphaMet reduced the Fas trimerization to the background level (Fig. 3a and 3c).
- Jurkat cells are one of the most widely used cell lines in Fas- mediated apoptosis investigation due to their high sensitivity. It was determined that these cells express very low levels of Met as evident by Western blot when compared to hepatic cells such as Hepa 1-6.
- the major form of Met observed in Jurkat cells was a pi 10 size form as opposed to the well known pi 45 form determined by two different antibodies against Met beta chain (c28, a polyclonal antibody that recognizes the C-terminal end of the beta chain, and DL-21, a monoclonal antibody that recognizes the extracellular portion of the beta chain).
- Flow cytometry analyses of the Propidium Iodide stained Jurkat cells indicated the sub-Gl population was reduced to 8% in the Fas-ligand with AlphaMet group, compared to the 48% apoptotic proportion in the Fas- ligand treated control.
- the YLGA motif mutated recombinant AlphaMet protein did not have any effects on the rate of apoptosis.
- the synthetic YLGA polypeptide blocked the Fas ligand induced apoptosis efficiently (see Fig. 4a) which is consistent with other binding experiments.
- recombinant AlphaMet protein and YLGA containing peptides (12mer, HHIYLGATNYIY, SEQ ID NO:26; and 4mer, YLGA, SEQ ID NO:l) significantly inhibited FasL induced apoptosis in these cells while mutated YLGA motif had no effect.
- Activation of caspase-3 was also inhibited by the wild type but not the YLGA mutated AlphaMet (Fig. 4d).
- apoptosis induced by the Fas agonist Jo2 was also efficiently inhibited by the addition of exogenous recombinant wild type AlphaMet.
- Stable cell lines form Hepa 1 -6 engineered to express AlphaMet also exhibited dramatic resistance to Fas killing as compared to control vector transfected clones. The results were consistent with those from the Jurkat cells (Fig. 4d). Since the YLGA (SEQ ID NO:l) motif did not block the binding of Jo2 to Fas, yet protected the Hepa 1-6 cells from Jo2 induced apoptosis, it is conceivable that the binding of Fas to AlphaMet or YLGA polypeptide blocks Fas trimerization and clustering, thus Fas activation. In support of this notion, reduced Jo2-induced Fas capping by AlphaMet and 12mer YLGA was observed (see Fig. 3f).
- Example 5 [0054] This example demonstrates that inventive polypeptides protect against apoptosis in vivo.
- mice that were injected with Jo2 only or Jo2 plus mutated AlphaMet showed massive hemorrhagic livers, which was evident grossly as well as microscopically, whereas the AlphaMet treated or the YLGA polypeptide treated animals showed little or no sign of hemorrhage and their livers were normal in appearance.
- Arakaki et al. "Hepatocyte growth factor/scatter factor activates the apoptosis signaling pathway by increasing caspase-3 activity in sarcoma 180 cells", Biochem Biophys Res Commun., 245(1), 211-215 (1998).
- Arakaki et al. "Involvement of oxidative stress in tumor cytotoxic activity of hepatocyte growth factor/scatter factor", J Biol Chem, 274(19), 13541-13546. (1999).
- Banner et al. "Crystal structure of the soluble human 55 kd TNF receptor-human TNF beta complex: implications for TNF receptor activation", Cell, 73(3), 431-445 (1993).
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2007064997A2 (en) * | 2005-12-01 | 2007-06-07 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Compounds and methods for inhibiting apoptosis |
WO2010102052A2 (en) | 2009-03-03 | 2010-09-10 | The Regents Of The University Of Michigan | Methods of inhibiting photoreceptor apoptosis |
EP2948178A4 (en) * | 2013-01-25 | 2016-07-20 | Thymon Llc | Compositions for selective reduction of circulating bioactive soluble tnf and methods for treating tnf-mediated disease |
WO2019246317A1 (en) | 2018-06-20 | 2019-12-26 | Progenity, Inc. | Treatment of a disease or condition in a tissue originating from the endoderm |
WO2020106754A1 (en) | 2018-11-19 | 2020-05-28 | Progenity, Inc. | Methods and devices for treating a disease with biotherapeutics |
WO2021119482A1 (en) | 2019-12-13 | 2021-06-17 | Progenity, Inc. | Ingestible device for delivery of therapeutic agent to the gastrointestinal tract |
EP4252629A2 (en) | 2016-12-07 | 2023-10-04 | Biora Therapeutics, Inc. | Gastrointestinal tract detection methods, devices and systems |
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WO2011066284A1 (en) * | 2009-11-25 | 2011-06-03 | The University Of North Carolina At Chapel Hill | Methods and compositions for the treatment of immune disorders |
MX2017014075A (en) | 2015-05-01 | 2018-07-06 | Onl Therapeutics Inc | Peptide compositions and methods of use. |
WO2019246141A1 (en) * | 2018-06-19 | 2019-12-26 | Cella Therapeutics, Llc | DRUG DELIVERY SYSTEMS COMPRISING A NEUROTROPHIC AGENT, AN APOPTOSIS SIGNALING FRAGMENT INHIBITOR (FAS) OR FAS LIGAND (FASL) INHIBITOR, A TUMOR NECROSIS FACTOR-α (TNF-α) OR TNF RECEPTOR INHIBITOR, A MITOCHONDRIAL PEPTIDE, AN OLIGONUCLEOTIDE, A CHEMOKINE INHIBITOR, OR A CYSTEINE-ASPARTIC PROTEASE |
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WO2005035564A2 (en) * | 2003-10-10 | 2005-04-21 | Xencor, Inc. | Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders |
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US6262239B1 (en) * | 1989-05-18 | 2001-07-17 | Yeda Research And Development Co., Ltd. | TNF receptor-specific antibodies |
US5430137A (en) * | 1989-10-25 | 1995-07-04 | Mycogen Corporation | Probes for the identification of Bacillus thuringiensis endotoxin genes |
FR2737209B1 (en) * | 1995-07-25 | 1997-09-19 | Bio Merieux | PEPTIDE CAPABLE OF BEING RECOGNIZED BY ANTIBODIES RECOGNIZING THE C33 ANTIGEN OF HEPATITIS C VIRUS |
US6001962A (en) * | 1996-11-15 | 1999-12-14 | The Regents Of The University Of California | Modified Fas ligands |
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WO2007064997A2 (en) * | 2005-12-01 | 2007-06-07 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Compounds and methods for inhibiting apoptosis |
WO2007064997A3 (en) * | 2005-12-01 | 2007-12-21 | Univ Pittsburgh | Compounds and methods for inhibiting apoptosis |
WO2010102052A2 (en) | 2009-03-03 | 2010-09-10 | The Regents Of The University Of Michigan | Methods of inhibiting photoreceptor apoptosis |
EP2403937A2 (en) * | 2009-03-03 | 2012-01-11 | The Regents of the University of Michigan | Methods of inhibiting photoreceptor apoptosis |
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EP2403937A4 (en) * | 2009-03-03 | 2013-04-10 | Univ Michigan | Methods of inhibiting photoreceptor apoptosis |
US8796223B2 (en) | 2009-03-03 | 2014-08-05 | The Regents Of The University Of Michigan | Methods of inhibiting photoreceptor apoptosis |
US9580502B2 (en) | 2013-01-25 | 2017-02-28 | Thymon, Llc | Compositions for selective reduction of circulating bioactive soluble TNF and methods for treating TNF-mediated disease |
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WO2019246317A1 (en) | 2018-06-20 | 2019-12-26 | Progenity, Inc. | Treatment of a disease or condition in a tissue originating from the endoderm |
WO2020106754A1 (en) | 2018-11-19 | 2020-05-28 | Progenity, Inc. | Methods and devices for treating a disease with biotherapeutics |
WO2020106757A1 (en) | 2018-11-19 | 2020-05-28 | Progenity, Inc. | Ingestible device for delivery of therapeutic agent to the gastrointestinal tract |
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EP4309722A2 (en) | 2019-12-13 | 2024-01-24 | Biora Therapeutics, Inc. | Ingestible device for delivery of therapeutic agent to the gastrointestinal tract |
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US20070184522A1 (en) | 2007-08-09 |
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WO2005117940A9 (en) | 2006-08-03 |
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